CN108559759A - Ternary shuttle vector and the method for building CLBV infectious clones using it - Google Patents

Abstract

The invention discloses a kind of ternary shuttle vector pCY, nucleotide sequence such as SEQ ID NO:Shown in 17.Also disclose a kind of method of structure CLBV infectious clones：(1) extraction infection CLBV plant total serum IgEs, PCR amplification is carried out after reverse transcription respectively using pCY CLBV1F, CLBV1R and CLBV2F, pCY CLBV2R, obtains specific fragment CLBV1, CLBV2 of covering CLBV full-length genomes；(2) Stu I, Sma I digestions pCY are used；(3) TAR is cloned：Using Li-acetate method by CLBV1, CLBV2 and linearisation pCY cotransformation saccharomycete YPH501, CLBV full length cDNA clone pCY CLBV are obtained by homologous recombination；(4) pCY CLBV plasmids convert Agrobacterium, are inoculated with citrus or this life cigarette, and identification obtains CLBV infectious clones.

Description

Ternary shuttle vector and the method for building CLBV infectious clones using it

Technical field

The invention belongs to molecular biology field, it is related to a kind of ternary shuttle vector and builds CLBV infectivities gram using its Grand method.

Background technology

Citrus leaf mottle viral (Citrus leaf blotch virus, CLBV) belongs to B-mode closterovirus section (Betaflexiviridae) line-up of delegates of citrus Tobamovirus (Citrivirus) can infect most of mandarin orange by grafting Tangerine kind can also have certain spread and epidemic risk by seed dispersal.Structure CLBV infectious clones will be helpful to Its molecular characterization and pathogenesis are solved, is also played an important roll for the popular prevention and control of CLBV.In addition, CLBV is in most citrus product It will not cause apparent viral infection symptoms on kind, be expected to be developed into the viral vectors with wide application prospect.CLBV bases Because a group size is about 8.7kb, including three open reading frame (Open reading frame, ORF), it is related to be separately encoded duplication Albumen, motor protein and coat protein, wherein motor protein are silencing suppressor, and there are methylated cap minor structure, 3 '-ends in 5 '-ends There are poly A tails.

Infectious clone is not only that virus research provides the single and reliable genetic stocks of background, it may also be used for research virus Movement, duplication and its pathogenesis, while also be further investigation virus with host's Coupling effects, carry out viral vectors transformation Solid foundation is established with application.However, the constructing technology of virus full length cDNA infectious clones is stronger, construction work difficulty Greatly.Some researches show that when building larger geneome RNA virus full length cDNA clone using Escherichia coli, due to its own coding Virus protein may to host strain generate toxic effect, so as to cause non-specificity recombinate, wild effect occur.This at The greatest difficulty faced for viral infectivity clone's structure and challenge.Virus full length genomic clone present in E.coli not The mechanism of stabilization is still unclear.Usually solved using following method：It is short again before infecting by virus sequence cDNA clones Temporary connection；Or utilize the copy lower carrier of number；Or the environment (such as reducing cultivation temperature) of regulation and control bacterial growth comes Reduce toxicity；Also have and have virose albumen using in insertion introne to viral whole genome sequence to avoid generating.But this A little methods take time and effort and success rate is low.

The adjoint homologous recombination of yeast conversion (TAR) is a kind of to realize multiple phases using the efficient homologous recombination system of yeast Mutually there are the DNA fragmentation assemble methods of homologous sequence.(Youssef F, Marais A, the Faure C, Gentit such as Youssef P,Candresse T.Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs:Apple chlorotic leaf spot virus as a case study.Virology Journal,2011,8(1):1-12) in structure apple chlorotic leaf spot virus (Apple chlorotic Leaf spot virus, ACLSV) when, from 36 through only identifying 1 in the correct E.coli clones of digestion with restriction enzyme A clone for having infectivity, shows to be cloned in E.coli cells by ACLSV that there is unstability, but obtains weight through yeast TAR technologies After group clone, the infectious clone of stable ACLSV is directly obtained by agriculture bacillus mediated inoculation.Arm spread moral wealth etc. (arm spread moral wealth, Shen Wentao, Yan Pu, Li little Ying, the yeast homologous recombination system of all E.coli-Free of roc a kind of are stablized rapid build PLDMV and are invaded The new method tropical crops journals of metachromia clone, 2017,38 (8):1492-1500) in structure papaya deformity mosaic virus When (Papaya leaf distortion mosaic virus, PLDMV), it is found that there is also shakinesses in Escherichia coli by PLDMV Determine phenomenon, and when being inserted into introne in the P3 genes in PLDMV, it is correct to obtain sequencing by Transformed E .coli after yeast recombinates Stabilization infectious clone.Since CLBV genomes are larger, its infectious clone is built using traditional digestion connection method, is taken When laborious and success rate it is low.In the trial of early period, the infectious clone for being difficult acquisition CLBV as host using Escherichia coli is guessed Survey may be related with aforementioned unstability.

Invention content

It is an object of the invention in view of the above technical problems, provide a kind of ternary shuttle vector and build CLBV using it The method of infectious clone.

The present invention realizes that the technical solution of its purpose is：

A kind of ternary shuttle vector pCY, nucleotide sequence such as SEQ ID NO:Shown in 17.

A method of structure CLBV infectious clones include the following steps：

(1) total serum IgE of extraction infection CLBV plant, using cDNA as template after reverse transcription, using two pairs of special primers PCY-CLBV1F, CLBV1R and CLBV2F, pCY-CLBV2R carry out PCR amplification respectively, obtain covering CLBV full-length genomes Two specific fragments CLBV1, CLBV2；The sequence of described primer pCY-CLBV1F, CLBV1R, CLBV2F, pCY-CLBV2R are distinguished Such as SEQ ID NO:Shown in 3~6；

(2) restriction enzyme Stu I, Sma I digestions such as SEQ ID NO are used:Ternary shuttle vector shown in 17 PCY obtains the carrier of pCY linearisations；

(3) TAR is cloned：Ternary shuttle vector pCY cotransformations by CLBV1, CLBV2 and are linearized using lithium acetate transformation method Saccharomycete YPH501 obtains CLBV full length cDNA clones pCY-CLBV by homologous recombination；

(4) infectious clone is identified：By pCY-CLBV plasmids by electroporated Agrobacterium C58C1, it is inoculated with citrus or sheet Raw cigarette, screening obtain CLBV infectious clones.

In the above-mentioned technical solutions, the construction method of the ternary shuttle vector pCY described in step (2) is：

A, using restriction enzyme Sac II single endonuclease digestion binary vector plasmid DK1317-2, the carrier linearized；

B, withPYES1L Vector are template, with SEQ ID NO:PYES2117F shown in 1~2, PYES2117R is that primer carries out PCR amplification, then recycles target fragment；

C, the recycling segment that DK1317-2 carriers and step B are obtained will be linearized and carries out recombination fusion, convert Escherichia coli JM109, select positive colony to get.

The beneficial effects of the invention are as follows：

The ternary shuttle vector pCY that the present invention is built, can not only pass through homologous recombination quick clone in yeast cells Virus full length cDNA, and amplification can also be replicated in Escherichia coli through agriculture bacillus mediated direct inoculation host plant； The present invention builds CLBV full length cDNA clones using the efficient homologous recombination system of yeast based on pCY, effectively overcomes The limitation of restriction enzyme site, entire regrouping process only need a yeast conversion, can be obtained in 2 weeks in traditional digestion cascade synthesis Viral full length cDNA clone is obtained, speed is fast, efficient；The invention is by TAR technologies by Corticovirus Genomic full_length cDNA Segment does not convert Escherichia coli with after ternary shuttle expression carrier pCY homologous recombinations, but directly converts Agrobacterium, to obtain Viral infectious clone, effectively overcome full length viral genome cDNA and generate toxicity in Escherichia coli or unstable cause The phenomenon that clone's failure, infectious clone pick-up rate significantly improves, up to 70% or so.

Description of the drawings

Fig. 1 is the plasmid map of shuttle vector pCY.

Fig. 2 is the systematic evolution tree of CLBV full-length genome nucleotide.

Fig. 3 is the RT-PCR testing results that CLBV infects this life cigarette, wherein M：DNA molecular standard, 1：Water, 2：It is negative right According to 3-18：Inoculation this life cigarettes of pCY-CLBV 1~16,19：Positive control.

Fig. 4 is Northern blot analysis CLBV geneome RNAs, wherein 1：Normal healthy controls, 2-6：Agriculture bacillus mediated PCY-CLBV 1,2,3,14,15 infects this life cigarette sample.

Fig. 5 is the symptom figure that agriculture bacillus mediated PCY-PVX is inoculated with after this life cigarette.

Fig. 6 is the RT-PCR testing results of PVX, wherein M：DL2000 DNA Marker；1~2：Sample；3：It is positive right According to；4：Negative control.

Specific implementation mode

Material therefor and reagent source of the present invention are as follows：

Yeast strain YPH501, agrobacterium strains C58C1 are given by French Thierry professors Candresse.

Restriction enzyme Sac II, Stu I, Sma I are purchased from Beijing NEB companies；LA Taqase、PrimerSTAR Max Premix、In-Fusion HD Cloning Kit、PrimeScript^TMII 1st Strand cDNA Synthesis Kit, JM109 are purchased from Dalian TaKaRa companies；Trizol reagents,PYES1LVector is purchased from the U.S. Invitrogen companies.

According to CLBV whole genome sequences (GenBank accession number is AJ318061) have been reported in NCBI, utilize 5.0 Software for Design of PrimerPriemer expands the special primer of CLBV gene orders.PCY-CLBV1F and CLBV1R, CLBV2F With pCY-CLBV2R two to special primer be respectively used for amplifying covering CLBV full-length genomes two specific fragment CLBV1, CLBV2；PYES2117F and PYES2117R is for expanding the segment for including yeast replication origin；PCY-PVX-F and pCY- PVX-R is for expanding PVX overall lengths to verify the validity of shuttle vector.In design of primers, CLBV1R and CLBV2F, pCY- CLBV1F is carried with the pCY after pCY carriers, pCY-PVX-R and the digestion after pCY carriers, pCY-CLBV2R and the digestion after digestion The overlap for all having 25~30bp between pCY carriers after body, pCY-PVX-F and digestion respectively, in order to complete in yeast At homologous recombination.Primer is synthesized (table 1) by English fine horse (Shanghai) Bioisystech Co., Ltd.

1 design of primers of table and its sequence

Note：It is SacII restriction endonuclease recognition sequences in box, underscore part is the 30bp homologous with pCY carriers.

Embodiment 1 builds ternary shuttle vector pCY

Structure binary vector plasmid DK1317-2 first：It is transformed to obtain with plasmid PCMBIA1301 and pXT1, PCMBIA1301 is commercially obtained, and pXT1 carriers are given by Agricultural University Of Nanjing professor Tao little Rong.It is with pXT1 carriers Template includes pXT1 gene expressions component (LB-2x35S-MCS-HDVRZ-NOS-RB) with primer TL1310F/TL1310R amplifications Segment.PCAMBIA1301 plasmids are digested with PvuI.Then with gel extraction kit to the amplification comprising expected segment and enzyme It cuts product and is purified and merged recombination.The VspI digestions of obtained fusion plasmid, big segment reconnect with structure PCAMBI-2x35S-MCS-HDVRZ-NOS, i.e. DK1317-2.

Using restriction enzyme Sac II single endonuclease digestion binary vector plasmid DK1317-2, the carrier linearized.Enzyme Cut reaction system：2.5 μ L, 10 × NEB Buffer of plasmid DK1317-2 12 μ L, restriction enzyme Sac II, 5 μ L, double steamings 23 μ L of water.Endonuclease reaction condition：37 DEG C of reaction 0.5h.

WithPYES1L Vector are template, and PYES2117F, PYES2117R are that primer amplification contains yeast The segment pYES1L-2117 of related replication origin：Reaction volume is 25 μ L, including distilled water 8.5 μ L, PrimerSTAR Max Premix (2 ×) 12.5 μ L, specific each 1 μ L of upstream and downstream primer, templatepYES1L Vector 1μL。 Reaction condition：98 DEG C of 1min, 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 2min, 30 cycles；72 DEG C of 5min, 4 DEG C of preservations.Then it utilizes DNA gel purification kit recycles target fragment, is named as pYES1L-2117.

It is recombinated using In-Fusion HD Cloning Kit, system includes：Linearisation DK1317-2 carriers add 2 μ L, Insert Fragment pYES1L-2117 add 4 μ L, In-Fusion Enzyme to add 2 μ L, dd H₂O polishings are to 10 μ L.50 DEG C, water-bath 30min, 4 DEG C of preservations.E. coli jm109 is converted, 37 DEG C are incubated overnight, and select positive colony, and acquisition can be in yeast-agriculture bar The ternary shuttle vector pCY grown in bacterium-Escherichia coli (its plasmid map is shown in Fig. 1).PCY contains yeast (ARS4/CEN5), root The reproduction element of cancer agrobacterium (pVS1 or IV) and Escherichia coli (pBR322ori), expression casette 2x35S-MCS- HDVRZ-NOS is between T-DNA insertion elements left arm and right arm.Therefore, carrier pCY can be used for DNA pieces in yeast cells Section homologous assembling and then recombinant DNA molecules are transformed into Agrobacterium or Escherichia coli.In addition, working as virus full length cDNA When being cloned between StuI the and SmaI restriction enzyme sites of pCY, expression casette 2x35S-MCS-HDVRZ-NOS will ensure that Virus genomic accurate starting and termination.The nucleotide sequence of ternary shuttle vector pCY is shown in SEQ ID NO:17.

Embodiment 2 builds the infectious clone of CLBV

One, the extraction of infected leaves total serum IgE is expanded with the segmentation of CLBV

It is complete to detect RNA by agarose gel electrophoresis for the total serum IgE that infected leaves is extracted according to Trizol reagent specifications Property.Using the total serum IgE extracted as masterplate, PrimeScript is used^TMII 1st Strand cDNA Synthesis Kit are closed At the first chain of cDNA.Using the cDNA as template, with two couples of special primer pCY-CLBV1F, CLBV1R and CLBV2F, pCY- CLBV2R obtains two specific fragments CLBV1, CLBV2 of covering CLBV full-length genomes, size to carrying out PCR amplification respectively Respectively 4500bp and 4247bp.PCR reaction systems are that 25 μ L include：8.5 μ L, PrimerSTAR Max Premix of distilled water (2 ×) 12.5 μ L, specific each 1 μ L of upstream and downstream primer, 1 μ L of template.Reaction condition：98 DEG C of 1min, 98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 2min, 35 cycles；72 DEG C of 5min, 4 DEG C of preservations.Then DNA gel purification kit recycling target fragment CLBV1 is utilized And CLBV2.

Two, yeast homologous recombination to construct CLBV full length cDNA clones

1) restriction enzyme Stu I, Sma I digested plasmid pCY are used, the carrier of pCY linearisations is obtained.Reactant System：1.0 μ L, 10 × NEB Buffer of plasmid pCY 13 μ L, restriction endonuclease sma I 5 μ L, 31 μ L of distilled water.Digestion is anti- Answer condition：Then 25 DEG C of reaction 0.5h add Stu I 1.0 μ L, 37 DEG C of incubation 0.5h.

2) lithium acetate transformation method transformed yeast and bacterium colony PCR are utilized

With reference to Zhao Guangyuan (Tuo D, ShenW, Yan P, Li X, Zhou P.Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.Viruses,2015,7(12):6241-6250) and Thierry Candresse (Youssef F,Marais A,Faure C,Gentit P,Candresse T.Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs:Apple chlorotic leaf spot virus as a case study.Virology Journal,2011,8(1):1-12) adopt Lithium acetate transformation method, in the centrifuge tube for taking the 100 μ L to 2mL of competent yeast cells YPH501 prepared, in order plus Enter following reagent：36 μ L of LiAc of PEG4000 (50%w/v) 240 μ L, 1mol/L, the 25 μ L of salmon sperm dna of 10mg/mL and Each 100ng of carrier 200ng, CLBV1, CLBV2 of pCY linearisations, concussion makes the component in transformation system mix well, at 30 DEG C 250r/min shakes bacterium 30min, 42 DEG C of water-bath heat shock 15min in shaking table；6000r/min centrifuges 3min, discards supernatant liquid, uses 300μL ddH₂Cell is resuspended in O, is uniformly coated in Trp deficiency screening flat boards, and 2~4d is cultivated in 30 DEG C.Picking monoclonal Bacterium colony carries out bacterium colony PCR identifications using special primer CLBV1F, CLBV5R.The result shows that in 24 bacterium colonies of picking 22 be PCR is positive.

3) CLBV full length cDNA clones are identified

The yeast plasmid for extracting aforementioned PCR positives bacterium colony, utilizes pCY-CLBV1F, CLBV1R and CLBV2F, pCY- CLBV2R carries out PCR detections to identify CLBV full length cDNA clones respectively, and 16 overall length plasmids are as a result identified from 22, CLBV full length cDNA clones positive rate 71.4%.It is random to select 1 full length cDNA clone sequencing, the results showed that：CLBV overall length sizes 8747bp, including 3 open reading frame, there are methyl in ORF1 5889nt, ORF2 1089nt, the ends ORF3 1092nt, 5' Change cap sequence, the ends 3' have poly (A).Sequence alignment result shows, the sequence and the listed other CLBV of GenBank The nucleotide identity of full length sequence is 79%~98%, wherein the EU857540 consistency highests with citrus source, are 98%, Consistency with JN983454, JN983455, JN983456 and the JN900477 in Kiwi berry source is 79%.It is utilizing On the phylogenetic tree of MEGA6 software buildings, the sequence and the CLBV separation strains in citrus source are gathered for cluster (Fig. 2).

Three, the infectivity identification of CLBV full length cDNA clones

1) Agrobacterium and inoculation are converted

CLBV full length cDNA clone plasmids are extracted, electroporated Agrobacterium C58C1 is passed through.Picking CLBV is positive single Clone's inoculation contains 20mgL^-1Rif, 50mgL^-1The LB liquid medium of Kan, 200rmin^-1, 28 DEG C of shaken cultivations 12~ 16h.Meanwhile being inoculated with the Agrobacterium monoclonal of Hc-Pro or P19 expression plasmids.Thalline were collected by centrifugation, with containing 10mmolL^{- 1}MgCl₂, 10mmolL^-1MES, 200 μm of olL^-1The inoculation buffer solution of AS suspends, and makes its OD₆₀₀Respectively 0.8~1.2 He 0.2~0.5.By this life Tobacco Leaves of 4~6 leaf phase of injection inoculation after standing 2h, and passes through vacuum immersion and be inoculated with 5~7 -day-old Brocade orange seedling.Meanwhile using the Agrobacterium of pCY empty plasmids as negative control.

2) RT-PCR methods detect CLBV

After being inoculated with 20d, the total serum IgE of the extraction upper blade of inoculation tobacco or newborn brocade orange blade, with special primer CLBV1F, CLBV5R carries out RT-PCR augmentation detections.PCR reaction systems are as follows：Template 1 μ L and ddH are successively added in PCR pipe₂1 μ L of O, 94 DEG C of unwinding 3min are placed on ice, and ddH is added₂2.3 μ L, 2 × 1step buffer of O, 5 0.2 μ L of μ L, CLBV1F, 0.2 μ L, PrimeScript 1step Enzyme Mix of CLBV5R, 0.3 μ L.Response procedures：50 DEG C of 30min, 94 DEG C of 2min, 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 45s, 35 cycles, 72 DEG C of 5min, 4 DEG C of stoppings.The result shows that pCY-CLBV1,2,3,5,8, 9,12,13,14,15,16 inoculation plant detect CLBV specific bands (as shown in Figure 3).Preliminary explanation, CLBV infectivities gram It is grand to build successfully.

3) Northern blot methods detect CLBV

It has been reported that and shows that this life cigarette inoculation CLBV does not show manifest symptom.To further determine that invading for constructed clone Positive this life cigarette sample of RT-PCR detections is randomly selected 5 progress Northernblot hybridization verifications by metachromia.

The preparation of probe and label：Probe is prepared using PCR, system is as follows：PCR buffer with MgCl₂5 μ L, 5 0.5 0.5 μ L, Enzyme mix of μ L, 01018-R of μ L, 01018-F of PCR DIG Probe Synthesis Mix, 0.75 μ L, template are sample CLBV reverse transcriptions cDNA 1 μ L, H₂O to 50μL.Response procedures：94 DEG C of 5min of pre-degeneration are denaturalized 94 DEG C 30s, anneal 53 DEG C of 30s, extends 72 DEG C of 30s, 35 cycles；Re-extend 72 DEG C of 5min, agarose gel electrophoresis testing result, It is saved backup after glue recycling.

Film preparation, hybridization and signal detection：Prepare 1% denaturing formaldehyde gel electrophoresis, 25v constant pressures, 4 DEG C, electrophoresis it is overnight.Again DNA in gel is gone into nylon membrane.Prehybridization：10.0mL DIG Easy Hyb are taken, are added in hybrid pipe, 50 DEG C of hybrid heaters Middle prehybridization 2h；Prehybridization solution is drained, the probe being newly denaturalized, mixing, 50 DEG C of hybridization are added in 10.0mL DIG Easy Hyb Hybridized overnight in instrument.Then film is washed, finally detects Northern blot hybridization signals with gel imaging system scanning nylon membrane.

As a result it shows：PCY-CLBV 1,2,3,14,15, which connects this life cigarette, can detect CLBV specific bands, and compare Any band (see Fig. 4) is not detected in sample, shows that pCY-CLBV 1,2,3,14,15 is infectious clone.

Brocade orange is inoculated with using vacuum infiltration method in addition, randomly selecting 5 and infecting the monoclonal that this life cigarette is positive (C.sinensis) seedling.After inoculation 40 days, CLBV specific bands can be detected in new foliation piece using RT-PCR, This further confirms that CLBV infectious clones are built successfully.

3 shuttle vector pCY of embodiment is built for PVX infectious clones

The shuttle vector pCY of the present invention is used for PVX (Potato Virus X) infectivity gram with reference to the method for embodiment 2 The amplification of grand structure, PVX full-length cDNAs uses primer pCY-PVXF, pCY-PVXR, yeast colony PCR detection to use primer PVX- F、PVX-R.It will be verified as positive pCY-PVX plasmids conversion Agrobacterium C58C1 and by Agrobacterium injection wetting this life cigarette, connect Observation result of the kind after 10 days is shown：Inject pCY-PVX tobacco symptom and positive control it is just the same, with negative control shape At apparent comparison (as shown in Figure 5).This illustrates that PVX has obtained high efficient expression, the yeast recombination based on ternary shuttle vector pCY Clone's system is built into work(, can be used for the infectious clone structure of other viruses.Acquire this life cigarette tender leaf after being inoculated with 10 days, profit Conventional RT-PCR detection is carried out with Plus methods extracting blade total nucleic acid.The results show that the upper blade of pCY-PVX inoculations amplifies The purpose band (as shown in Figure 6) being consistent with expected size, this yeast recombinant clone body of explanation based on ternary shuttle vector pCY System builds successfully.

4 shuttle vector pCY of embodiment is built for CYVCV infectious clones

With reference to embodiment 2 method by the present invention shuttle vector pCY for CYVCV (Citrus Yellowing vein clearing virus, Citrus yellow clearing vein virus) infectious clone structure.By TAR technologies by its full length cDNA clone To ternary carrier pCY, Citrus Yellowing vein clearing virus Anyue Sichuan isolate full length cDNA clone, sequencing knot are successfully obtained Fruit shows that CYVCV-AY genomes are 7529bp, including 6 open reading frame, consistent with the CYVCV separation strains reported at present. Sequence alignment result shows that the nucleotide sequence similarity of 4 randomly selected CYVCV-AY full length cDNA clones is equal 99% or more；On systematic evolution tree, 4 clones of CYVCV-AY gather with the CYVCV separation strains in citrus source for cluster. At home and abroad obtain the infectious cDNA clone of CYVCV, Symptom Observation and RT-PCR testing result tables for the first time by TAR clones Bright, the pCY-CYVCV obtained has infectivity.

Sequence table

<110>Southwestern University

<120>Ternary shuttle vector and the method for building CLBV infectious clones using it

<160> 17

<210> 1

<211> 39

<212> DNA

<213>Artificial sequence

<223> PYES2117F

<400> 1

gccgattttg aaaccgcgga gtcagtgagc gaggaagcg 39

<210> 2

<211> 39

<212> DNA

<213>Artificial sequence

<223> PYES2117R

<400> 2

ctgcctgtga tcaccgcggc atcttttact ttcaccagc 39

<210> 3

<211> 59

<212> DNA

<213>Artificial sequence

<223> pCY-CLBV1F

<400> 3

atataaggaa gttcatttca tttggagagg agaaaagcaa cgaaagcaac ctacacaac 59

<210> 4

<211> 30

<212> DNA

<213>Artificial sequence

<223> CLBV1R

<400> 4

aaagggtcac cctccaacct ttcctcccta 30

<210> 5

<211> 30

<212> DNA

<213>Artificial sequence

<223> CLBV2F

<400> 5

tagggaggaa aggttggagg gtgacccttt 30

<210> 6

<211> 57

<212> DNA

<213>Artificial sequence

<223> pCY-CLBV2R

<400> 6

cgcgaggagg tggagatgcc atgccgaccc tttttttttt tttttttttt gtctaaa 57

<210> 7

<211> 56

<212> DNA

<213>Artificial sequence

<223> pCY-PVX-F

<400> 7

atataaggaa gttcatttca tttggagagg agaaaactaa accatacacc accaac 56

<210> 8

<211> 99

<212> DNA

<213>Artificial sequence

<223> pCY-PVX-R

<400> 8

cgcgaggagg tggagatgcc atgccgaccc gggttttttt tttttttttt tttttttttt 60

tttttttttt tttttttttt tttttttttt tttatttat 99

<210> 9

<211> 22

<212> DNA

<213>Artificial sequence

<223> CLBV1F

<400> 9

agccatagtt gaaccattcc tc 22

<210> 10

<211> 20

<212> DNA

<213>Artificial sequence

<223> CLBV5R

<400> 10

gcagatcatt caccacatgc 20

<210> 11

<211> 20

<212> DNA

<213>Artificial sequence

<223> PVX-F

<400> 11

atgtcagcac cagctagcac 20

<210> 12

<211> 23

<212> DNA

<213>Artificial sequence

<223> PVX-R

<400> 12

ggatccttat ggtggtggta gag 23

<210> 13

<211> 18

<212> DNA

<213>Artificial sequence

<223> 01018-F

<400> 13

aggtcttctt cgccattt 18

<210> 14

<211> 18

<212> DNA

<213>Artificial sequence

<223> 01018-R

<400> 14

cccttcttcc tccagttt 18

<210> 15

<211> 41

<212> DNA

<213>Artificial sequence

<223> TL1310F

<400> 15

aactcgagct tgtcgattaa tgcatgcctg cagtcaacat g 41

<210> 16

<211> 33

<212> DNA

<213>Artificial sequence

<223> TL1310R

<400> 16

aaatgtttga acgatcgggg aaattcgagc tct 33

<210> 17

<211> 10347

<212> DNA

<213>Artificial sequence

<223>Ternary shuttle vector pCY

<400> 17

attaatgcat gcctgcagtc aacatggtgg agcacgacac tctcgtctac tccaagaata 60

tcaaagatac agtctcagaa gaccagaggg ctattgagac ttttcaacaa agggtaatat 120

cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag 180

aaaaggaaga tggcttctac aaatgccatc attgcgataa aggaaaggct atcgttcaaa 240

gaatgcctct accgacagtg gtcccaaaga tggacccccc acccacgagg aacatcgtgg 300

aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat aacatggtgg 360

agcacgacac tctcgtctac tccaagaata tcaaagatac agtctcagaa gaccagaggg 420

ctattgagac tttcaacaaa gggtaatatc gggaaacctc ctcggattcc attgcccagc 480

tatctgtcac ttcatcgaaa ggacagtaga aaaggaagat ggcttctaca aatgccatca 540

ttgcgataaa ggaaaggcta tcgttcaaga atgcctctac cgacagtggt cccaaagatg 600

gacccccacc cacgaggaac atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 660

aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 720

cgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggcct atgagtaaag 780

gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg 840

ggtacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc 900

ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt 960

tctcttatgg tgttcaatgc ttttcaagat acccagatca tatgaagcgg cacgacttct 1020

tcaagagcgc catgcctgag ggatacgtgc aggagaggac catcttcttc aaggacgacg 1080

ggaactacaa gacacgtgct gaagtcaagt ttgagggaga caccctcgtc aacaggatcg 1140

agcttaaggg aatcgatttc aaggaggacg gaaacatcct cggccacaag ttggaataca 1200

actacaactt ccacaacgta tacatcatgg ccgacaagca aaagaacggc atcaaagcca 1260

acttcaagac ccgccacaac atcgaagacg gcggcgtgca actcgctgat cattatcaac 1320

aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtccacac 1380

aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg 1440

taacagctgc tgggattaca catggcatgg atgaactata caaataaccc gggtcggcat 1500

ggcatctcca cctcctcgcg gtccgacctg ggcatccgaa ggaggacgtc gtccactcgg 1560

atggctaagg gagagctcga atttccccga tcgttcaaac atttggcaat aaagtttctt 1620

aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt 1680

taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat 1740

tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta 1800

ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattaa actatcagtg 1860

tttgacagga tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataacgga 1920

tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac 1980

agggttcccc tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg 2040

gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta 2100

cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca 2160

taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg 2220

ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg 2280

ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc 2340

gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag 2400

tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca 2460

tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc 2520

cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa 2580

tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc 2640

cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag 2700

gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg 2760

cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg 2820

aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac 2880

aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga 2940

ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg 3000

gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt 3060

ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag 3120

cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg 3180

gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc 3240

gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc 3300

gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt 3360

gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc 3420

gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc 3480

gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg 3540

gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc 3600

gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg 3660

tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc 3720

gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag 3780

gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga 3840

gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa 3900

cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg 3960

aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc 4020

tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga 4080

attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg 4140

gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc 4200

cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc 4260

atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc 4320

cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca 4380

agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc 4440

gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga 4500

cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg 4560

tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc 4620

cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc 4680

ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt 4740

gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca 4800

gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg 4860

tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag 4920

ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc 4980

tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc 5040

cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc 5100

cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc 5160

cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc 5220

ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg 5280

caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca 5340

aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat 5400

tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta 5460

cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc 5520

cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact 5580

gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc 5640

cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg 5700

cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg 5760

gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac 5820

acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 5880

cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac 5940

gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag 6000

agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 6060

gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 6120

ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 6180

aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 6240

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6300

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6360

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6420

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 6480

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 6540

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 6600

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 6660

gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 6720

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 6780

cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 6840

tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 6900

tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt 6960

ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc 7020

gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc 7080

gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc 7140

tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc 7200

atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc 7260

ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt 7320

atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc 7380

gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc 7440

ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac 7500

aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt 7560

ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt 7620

atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag 7680

ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta 7740

cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc 7800

cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg 7860

gcgtataaca tagtatcgac ggagccgatt ttgaaaccgc ggagtcagtg agcgaggaag 7920

cgcgtaacta taacggtcct aaggtagcga atcctgatgc ggtattttct ccttacgcat 7980

ctgtgcggta tttcacaccg catagatcgg caagtgcaca aacaatactt aaataaatac 8040

tactcagtaa taacctattt cttagcattt ttgacgaaat ttgctatttt gttagagtct 8100

tttacaccat ttgtctccac acctccgctt acatcaacac caataacgcc atttaatcta 8160

agcgcatcac caacattttc tggcgtcagt ccaccagcta acataaaatg taagctttcg 8220

gggctctctt gccttccaac ccagtcagaa atcgagttcc aatccaaaag ttcacctgtc 8280

ccacctgctt ctgaatcaaa caagggaata aacgaatgag gtttctgtga agctgcactg 8340

agtagtatgt tgcagtcttt tggaaatacg agtcttttaa taactggcaa accgaggaac 8400

tcttggtatt cttgccacga ctcatctcca tgcagttgga cgatatcaat gccgtaatca 8460

ttgaccagag ccaaaacatc ctccttaagt tgattacgaa acacgccaac caagtatttc 8520

ggagtgcctg aactattttt atatgctttt acaagacttg aaattttcct tgcaataacc 8580

gggtcaattg ttctctttct attgggcaca catataatac ccagcaagtc agcatcggaa 8640

tctagagcac attctgcggc ctctgtgctc tgcaagccgc aaactttcac caatggacca 8700

gaactacctg tgaaattaat aacagacata ctccaagctg cctttgtgtg cttaatcacg 8760

tatactcacg tgctcaatag tcaccaatgc cctccctctt ggccctctcc ttttcttttt 8820

tcgaccgaat taattcttaa tcggcaaaaa aagaaaagct ccggatcaag attgtacgta 8880

aggtgacaag ctatttttca ataaagaata tcttccacta ctgccatctg gcgtcataac 8940

tgcaaagtac acatatatta cgatgctgtt ctattaaatg cttcctatat tatatatata 9000

gtaatgtcgt gatctatggt gcactctcag tacaatctgc tctgatgccg catagttaag 9060

ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 9120

atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 9180

gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 9240

tgtcatgata ataatggttt cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt 9300

atcttttaat gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg 9360

tatttggatt ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa 9420

ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac 9480

aagaaaagca gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca 9540

ggattttcgt gtgtggtctt ctacacagac aaggtgaaac aattcggcat taatacctga 9600

gagcaggaag agcaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca 9660

tcttcggaaa acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt 9720

tatatattta tattaaaaaa tttaaattat aattattttt atagcacgtg atgaaaagga 9780

cccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 9840

acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 9900

aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 9960

attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgccgcggt 10020

gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca 10080

tccgtgtttc aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag 10140

caaagtctgc cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc 10200

tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg 10260

caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac 10320

gtttttaatg tactgaatta acgccga 10347

Claims (3)

1. a kind of ternary shuttle vector pCY, which is characterized in that its nucleotide sequence such as SEQ ID NO:Shown in 17.

2. a kind of method of structure CLBV infectious clones, which is characterized in that include the following steps：

(1) total serum IgE of extraction infection CLBV plant, using cDNA as template after reverse transcription, using two couples of special primer pCY- CLBV1F, CLBV1R and CLBV2F, pCY-CLBV2R carry out PCR amplification respectively, obtain two of covering CLBV full-length genomes Specific fragment CLBV1, CLBV2；The sequence of described primer pCY-CLBV1F, CLBV1R, CLBV2F, pCY-CLBV2R are respectively such as SEQ ID NO:Shown in 3~6；

(2) restriction enzyme Stu I, Sma I digestions such as SEQ ID NO are used:Ternary shuttle vector pCY, obtains shown in 17 The carrier linearized to pCY；

(3) TAR is cloned：Ternary shuttle vector pCY cotransformation yeast by CLBV1, CLBV2 and is linearized using lithium acetate transformation method Bacterium YPH501 obtains CLBV full length cDNA clones pCY-CLBV by homologous recombination；

(4) infectious clone is identified：By pCY-CLBV plasmids by electroporated Agrobacterium C58C1, it is inoculated with citrus or this life cigarette, Identification obtains CLBV infectious clones.

3. the method for structure CLBV infectious clones as claimed in claim 2, which is characterized in that three described in step (2) The construction method of first shuttle vector pCY is：

A, using restriction enzyme SacII single endonuclease digestion binary vector plasmid DK1317-2, the carrier linearized；

B, with GenePYES1LVector is template, with SEQ ID NO:PYES2117F, PYES2117R shown in 1~2 are Primer carries out PCR amplification, then recycles target fragment；

C, the recycling segment that DK1317-2 carriers and step B are obtained will be linearized and carries out recombination fusion, convert Escherichia coli JM109, select positive colony to get.